Hepatoma polarization limits CD81 and hepatitis C virus dynamics.

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81(ΔC)) and its effect(s) on HCVpp mobility and infection. CD81(ΔC) showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81(ΔC) expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner.

Domain formation in models of the renal brush border membrane outer leaflet.

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.

Temperature dependence of the topology of supported dimirystoyl-distearoyl phosphatidylcholine bilayers.

Topology of fluid and gel domains in the supported bilayer two-component system formed from equimolar mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) was determined by AFM, at various temperatures corresponding to the gel and the gel + fluid region of the phase diagram. The data show that, in the disconnected fluid part of the DMPC/DSPC gel-liquid crystal-phase-separation region, the size of fluid domains markedly exceeds that predicted from spectroscopic experiments or from Monte Carlo simulations. They provide a direct evidence for the transition from the disconnected fluid to the disconnected gel region of the phase diagram, again with gel-phase domains much larger than expected. Finally, images of the gel phase at different temperatures suggest that structural rearrangements of the phospholipids can disrupt the continuity of the supported bilayer.

The lysine-rich C-terminal tail of heparin affin regulatory peptide is required for mitogenic and tumor formation activities.

Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two beta-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic.

Glycosaminoglycans promote HARP/PTN dimerization.

Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a secreted polypeptide which binds to heparin and plays a key role in cellular growth and differentiation. In order to assess the determinants potentially important to its biological activity, we tested the ability of HARP to oligomerize, a process involved in mitogenic activity of the heparin-binding fibroblast growth factor. Using dissuccinimidyl suberate cross-linking experiments and affinity chromatography, we report that human HARP forms noncovalent dimers. Dimerization is dependent on the presence of heparin or other sulfated glycosaminoglycans, as chlorate treatment of cells inhibits this process. In vitro, different glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate-C, also induce a dimer assembly of HARP. The relevance of this process was supported by experiments demonstrating that HARP is secreted as a dimer in conditioned medium of NIH-3T3 cells that overexpressed this growth factor and is also associated to the cell surface or to the extracellular matrix.

[Implication of HARP in angiogenesis and possible therapeutic role]. / Implication de l'HARP dans l'angiogenèse et rôle thérapeutique possible.

HARP (heparin affin regulatory peptide), also called pleiotrophin (PTN), belongs to the heparin binding growth factors (HBGFs) family. Several new data suggest a role for HARP during the various stages of angiogenesis. In vivo, HARP is localised in endothelial cells of blood capillaries. In vitro, HARP displays mitogenic activity on endothelial cells, induces the formation of capillary-like structures in collagen gel, and degrades extracellular matrix via stimulation of plasminogen activator activity. HARP is also involved in neoangiogenesis during tumor progression. This review discusses the possible role of HARP in tumor angiogenesis and its therapeutic implications.

Upregulation of the angiogenic factor heparin affin regulatory peptide by progesterone in rat uterus.

Heparin affin regulatory peptide (HARP), also named pleiotropin, is a secreted polypeptide that belongs to a new family of heparin-binding growth/differentiation factors. In this study, we investigated the expression and distribution of HARP mRNA and protein in rat uterus. Semi-quantitative reverse transcriptase PCR experiments showed variations in HARP mRNA levels throughout the estrous cycle, with a maximum during diestrus, pointing to hormonal regulation of HARP mRNA expression. Uterine expression of HARP mRNA was studied in ovariectomized animals treated with 17 beta-estradiol, progesterone alone or progesterone and RU486. In these experiments, progesterone upregulated HARP mRNA expression. Induction was observed 6 h after progesterone injection and was inhibited by RU486 treatment. In contrast, after 17 beta-estradiol injection, a slight decrease in HARP mRNA expression was observed. In situ hybridization studies with digoxigenin-labeled DNA probe revealed that HARP mRNA was present in smooth muscle cells of both myometrium and blood vessels and also in endothelial cells from endometrium. Immunohistochemical studies showed that HARP expression was not limited to cells that expressed HARP mRNA, but also occurred in both the luminal and glandular epithelium even though its transcript was never detected. We conclude that HARP may mediate the effects of progesterone on the homeostasis and vascularization of uterine tissue.

Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin.

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

Fate of a fluorescent inhibitor of endopeptidase-24.11 using enzyme-expressing MDCK cells. Modification of its cellular processing with a monoclonal antibody.

Neutral endopeptidase-24.11 (NEP) is a membrane-bound zinc metallopeptidase which cleaves biologically active peptides such as the enkephalins and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)-amino-1- hexyl]-thiocarbamide (FTI), the fate of the inhibitor-enzyme complex was investigated by videomicrofluorimetry using MDCK epithelial cells expressing the rabbit peptidase thanks to a retroviral expression vector. N-[3-(R,S)-[(hydroxyamino) carbonyl]-2-benzyl-1-oxopropyl]- glycine (HACBOGly) and the corresponding tritiated molecule were also used to measure the cellular pathway of inhibitor-NEP complexes. In the present paper, we demonstrate that, for short incubation times, the fluorescent probe preferentially labeled brush border membranes of the apical side of the MDCK cells. After more than 1 h incubation, a honeycomb pattern of fluorescence was observed in videomicrofluorimetry suggesting that part of the inhibitor was bound or localized close to the basolateral plasma membrane. Confocal experiments confirmed the transcytosis of FTI/NEP complex, from the apical to the basolateral domain. Using [3H]HACBOGly on filter-grown cells, after 2 and 4 h incubation at 37 degrees C, the percentage of basolateral membrane-bound molecules was estimated to be about 12 and 23%, respectively. The coincubation of the cells with FTI and 2B12, a monoclonal antibody raised against the rabbit enzyme, greatly modified the fluorescence pattern. A patchy fluorescence was observed for short incubation times, corresponding to cluster formation induced by antigen-antibody binding. For longer incubation times (> 1 h), in addition to the basolateral labeling, some intracellular fluorescent vesicles were observed essentially localized in the vicinity of the nucleus. The colocalization of FTI with Texas Red isothiocyanate-labeled Concanavalin A (TRITC-Con A) strongly suggests an endosomal/lysosomal internalization pathway when FTI was incubated in the presence of 2B12 mAb.

Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity.

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.

Rat endopeptidase-24.18 alpha subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells.

Endopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The alpha and beta subunits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 alpha subunit and to test the functionality of the astacin-like domain in the alpha subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat alpha subunit. Despite the presence of its putative transmembrane domain, the alpha subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the alpha subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the alpha subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Complete sequence of rabbit kidney aminopeptidase N and mRNA localization in rabbit kidney by in situ hybridization.

We have isolated and sequenced a full-length cDNA for rabbit kidney aminopeptidase N (APN). The 3-kilobase cDNA contains 12 nucleotides of the 5' noncoding region, a 2898 nucleotide long open reading frame, and 113 nucleotides of the 3' untranslated region. The open reading frame encodes a type II membrane protein of 966 amino acid residues composed of a 10 residue NH2-terminal cytosolic domain, a 23 residue transmembrane domain, and a large 933 residue ectodomain that contains the active site. Rabbit APN has eight potential N-glycosylation sites and seven cysteine residues, one of which is located in the transmembrane domain. Computer analysis showed that the enzymes from human, rat, and rabbit were highly conserved, except for the stalk region immediately downstream from the transmembrane domain. Using in situ hybridization techniques we showed that in rabbit kidney, APN mRNA is present in proximal tubules but not in glomeruli, which corresponds to the localization of the protein observed by immunohistochemistry. Taken together, our results strongly suggest that the expression of APN in kidney is modulated at mRNA levels and not at translational and (or) posttranslational levels.

Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.

Increase of neutral endopeptidase-24.11 with cellular density and enzyme modulation with an inhibitor on human Reh6 cell line.

Neutral endopeptidase (EC 3.4.24.11, NEP) is an ectoenzyme, identified as the common acute lymphoblastic leukemia antigen (CALLA, CD10). This enzyme is involved in the inactivation of regulatory peptides such as enkephalins and atrial natriuretic peptide and its expression on the cell surface is therefore essential. NEP levels have been measured under different conditions on leukemic cell lines. NEP activity per cell was found to increase during the cell growth of Reh6 and CEM cells, a cell-cell contact mechanism being suggested by experiments using Transwell cell chambers. The same process was not observed with ICIG-7 fibroblasts. The numbers of enzymatic sites was also found to be selectively modulated by treatment with 0.1 microM N-[3-(R,S)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]glycine (HACBOGly), a potent (Ki = 1.4 nM) and specific inhibitor of NEP. A maximal 13% decrease in sites was observed after 8 hr incubation, this effect disappearing after 12 hr. This weak but specific negative modulation was not observed with a compound, chemically related to HACBOGly, which has a 10,000-fold lower inhibitory potency. The modulation was inhibited by low temperature or monensin treatment and could be brought about by an internalization of the enzyme, compensated for by an increased biosynthesis or by the sequestration of NEP in a non-membranous compartment.

Effects of monoclonal antibodies raised against the common acute lymphoblastic leukemia antigen on endopeptidase-24.11 activity.

The common acute lymphoblastic leukemia antigen (CALLA, CD10) has been identified as neutral endopeptidase-24.11 (NEP), a mammalian ectoenzyme involved in the inactivation of regulatory peptides, such as the enkephalins and atrial natriuretic peptide. Twenty monoclonal antibodies directed against the human antigen, were tested for their ability to inhibit the enzymatic activity of the human and rat peptidases expressed by cell lines. Six anti-CALLA antibodies were found to inhibit 50% or more of the hydrolysis of D-Ala2-leucine enkephalin by the neutral endopeptidase present on the human leukemic cell line Reh6 and, to a lesser extent, the hydrolysis of atrial natriuretic peptide. This may indicate that their binding may affect regions of the active site more important for the dipeptidylcarboxypeptidase activity of the enzyme. Only four antibodies cross-reacted with the peptidase from the rat epithelial cell line Rat2, as shown by membrane immunofluorescence, and these also partially inhibited enzyme activity. No antibody was able to inhibit completely the activity of the human and rat enzymes and all the active antibodies appeared to behave as non-competitive inhibitors of substrate cleavage. These monoclonal antibodies could be used in mapping studies of NEP.